Cross-Talk Between Autophagy And Pyroptosis Of Pulmonary Microvascular Endothelial Cells Induced By Oxygen And Glucose Deprivation/Reoxygenation

Part I Role of pyroptosis induced by oxygen-glucose deprivation/reoxygenationin pulmonary microvascular endothelial cells injuryAIM:To explore whether oxygen-glucose deprivation/reoxygenation(OGD/R)causes pyroptosis of human pulmonary microvascular endothelial cells(HPMVECs)and its role in cell damage of HPMVECs.Methods:HPMVECswere used to replicate the OGD/R cell model for simulating the ischemia-reperfusion(I/R)process of pulmonary microvascular endothelial cells during cardiopulmonary bypass(CPB).HPMVECs were divided into control group,OGD/R group and VX-765 group.The cells in control group were cultured normally.The cells in OGD/R group were treated with oxygen-glucose deprivation for 8 h and recovery for 12 h.The cells in VX-765 group were treated with VX-765for4 h before OGD/R,and the remaining culture conditions were the same as those in OGD/R group.The m RNA and protein expression levels ofcysteinyl aspartate specific roteinase-1(caspase-1),nucleotidebinding oligomerization domain-like receptor protein 3(NLRP3),and apoptosis-associated speck-like protein containing CARD(ASC)were detected by RT-q PCR and Western blot.The levels of interleukin(IL)-1βand IL-18 in the supernatant were measured by ELISA.The lactate dehy drogenase(LDH)level in the supernatant was determined by lactate dehydrogenase kit.Results:Compared with control group,the m RNA and protein expression of caspase-1,NLRP3 and ASC was increased in OGD/R group(P<0.05),the secretion of inflammatory cytokines IL-1β and IL-18 were also increased(P<0.05).The up-regulation of m RNA and protein expression levels of caspase-1,NLRP3 and ASC,also the secretion of IL-1β and IL-18 caused by OGD/R were reversed by a caspase-1 inhibitor VX-765(P<0.05),and the LDH level after OGD/R was decreased by VX-765treatment(P<0.05).Conclusion:OGD/R causes pyroptosis of HPMVECs,and the use of inhibitor VX-765 can alleviate the damage caused by OGD/R to HPMVECs by inhibiting pyroptosis.Part II Changes of autophagy activity of pulmonary microvascular endothelial cells and its effect on pyroptosis under oxygen-glucose deprivation/reoxygenationAIM:To study the effect of OGD/R on autophagy of HPMVECs,and to explore the relationship between autophagy and pyroptosis under OGD/R conditions.Methods: HPMVECs were divided into control group,OGD/R group,Rapa group and 3-MA group.The control group and the OGD/R group were treated as before,the Rapa group and the 3-MA group were treated with rapamycin and 3-MA for 4 h before OGD/R treatment,respectively.The cell survival rate was determined by MTT assay.The protein expressions of the autophagy-related proteins LC3-II/I,Beclin-1,P62,and the pyroptosis-related proteins caspase-1,NLRP3,ASC were detected by Western blot.Meanwhile,the expression of NLRP3 in cells was observed by immunofluorescence technique.The expressions of inflammatory cytokines IL-1β and IL-18 were measured by ELISA.And the LDHlevel in the supernatant was determined by lactate dehydrogenase kit.Results:Compared with control group,the cell survival rate of OGD/R group was significantly decreased,and the expression of autophagy-related proteins LC3-II/I and Beclin-1 were down-regulated,and the expression of P62 was increased(P<0.05).Compared with the OGD/R group,after using the autophagy activator rapamycin,the expression of LC3-II/I and Beclin-1 was upregulated,the expression of P62 was decreased,the expression of pyroptosis-related proteins caspase-1,NLRP3 and ASC was decreased,and the survival rate of cells was increased(P<0.05);Compared with the OGD/R group,tthe expression of LC3-II/I and Beclin-1 was down-regulated,the expression of P62 was increased in the 3-MA group,and the expression of pyroptosis-related proteins caspase-1,NLRP3,and ASC was increased,the survival rate of cells was decreased(P<0.05);Immunofluorescence results showed that the intracellular expression of NLRP3 was decreased after the addition of the activator rapamycin,while the intracellular expression of NLRP3 was increased in 3-MA group(P<0.05).Compared with OGD/R group,the secretion of inflammatory cytokines IL-1β and IL-18 were decreased in RAPA group,and the LDH level was down-regulated,while the expression of inflammatory cytokines IL-1β and IL-18 were up-regulated in 3-MA group,and the LDH level in RAPA group was up-regulated(P<0.05).Conclusion: Autophagy can reduce cell damage by degrading NLRP3 inflammasome to inhibit the pyroptosis of HPMVECs caused by OGD/R.